Epstein-Barr virus-negative NK cell line

ABSTRACT

The present invention provides Epstein-Barr virus (EBV)-negative NK cell lines. The NK cell lines of the present invention are useful for screening factors associated with the proliferation and expression functions of NK cells and to discover factors produced by the NK cells. In addition, the cell lines are immortalized despite the fact that they are EBV-negative. Thus, unknown mechanisms of oncogenesis may be elucidated through an understanding of the mechanisms underlying the immortalization of the cell lines of the present invention.

TECHNICAL FIELD

The present invention relates to an NK cell line.

BACKGROUND ART

A natural killer cell (hereinafter abbreviated as “NK cell”) isactivated by interferon and interleukin-2 (hereinafter abbreviated as“IL2”), and the activated cell destroys tumor cells and such. While thebiological defense mechanism dependent on T cells and B cells functionsas a result of immune response, the NK cell-dependent defense mechanismworks via the activation by cytokines. Namely, the complicated processof immune response is not required for the defensive function of NKcells. Therefore, NK cells have been believed to play an important roleat the forefront of the biological defense mechanisms.

Thus, the biological defense mechanisms may be controlled by modifyingthe activation and proliferation of NK cells. However, many obscuritiesregarding the function of NK cells remain to be clarified.

On the other hand, NK cells are lymphocytes and form lymphomas upontumorigenic transformation. For example, primary nasal angiocentric NKlymphoma is a lymphoma found in Asian countries. Primary nasalangiocentric NK lymphomas are generally resistant to various anti-canceragents and often have a bad prognosis. Thus, elucidation of themechanism of NK cell tumorigenesis is an important object for developingtherapeutic methods against this disease.

Infection with Epstein-Barr virus (hereinafter abbreviated as “EBV”) hasbeen suggested to be involved in the development lymphoma. For example,using a cell line established from Burkitt's lymphoma (hereinafterabbreviated as “BL”), the influence of the EBV genome on B cell lymphomahas been reported (Journal of Virology 6069-6073, 1994, “Isolation ofEpstein-Barr Virus (EBV)-negative cell clones from the EBV-positiveBurkitt's lymphoma (BL) line Akata: malignant phenotypes of BL cell aredependent on EBV.”). According to this report, malignant phenotypes ofBL were induced in EBV-negative Akata cells by infection of EBV into thecells. However, there is no report describing the involvement of EBVwith NK cells. Thus, elucidation of the mechanism underlying thetumorigenesis of NK cells is desired in the art. In particular, once anEBV-negative NK cell line is established, such a cell line can serve asan important research tool for studying the effect of EBV on NK cells.

NK cell lines are useful for performing studies on NK cells, e.g.,studies on the regulation of activation and proliferation of NK cells,the mechanism underlying NK cell tumorigenesis, etc. When such a cellline is made available, a common cell can be easily shared by manyresearchers. Most importantly, the use of a cell line obviates the needto use fresh blood as an experimental tool, which would facilitate theperformance of such experiments.

The present inventors have already established a cell line NK-YS derivedfrom NK lymphoma (Blood 192: 1374-1383, 1998). The NK cell line NK-YS isan EBV-positive cell line. To assess the involvement of EBV, it isexpected to establish an EBV-negative cell line.

As described above, the establishment of an EBV-negative cell line froman Akata cell that is a B-cell lymphoma-derived cell line has beenreported. The elimination of EBV in B cells occurs due to long-termpassage of EBV-positive lymphoma-derived cells. This enabled theestablishment of an EBV-negative B cell line. However, there is no knowntechnique to establish EBV-negative NK cell lines.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide EBV-negative NK celllines and uses thereof.

To achieve the above-described objective, the present inventorsrepeatedly separated various NK cells and examined whether the cellswere infected with EBV. As a result, the present inventors successfullyestablished an EBV-negative cell line from NK cells derived from apatient with primary nasal lymphoma. The present inventors alsodiscovered that activities to control the NK cell activity can bedetected using this NK cell line; and thus, the present invention wascompleted. Specifically, the present invention provides cell lines,methods for detecting activities to control the NK cell activity usingthe cell lines, and screening method using the cell lines as follows.

[1] An Epstein-Barr virus-negative NK cell line.

[2] The cell line according to [1], which is derived from human.

[3] The cell line according to [1], which is derived from a leukemiacell.

[4] The cell line according to [3], wherein the leukemia is a lymphoma.

[5] The cell line according to [4], wherein the lymphoma is a nasalangiocentric lymphoma.

[6] The cell line according to [5], wherein the nasal angiocentriclymphoma is an NK lymphoma.

[7] The cell line according to [1], which is derived from anEpstein-Barr virus-positive host.

[8] The cell line according to [1], which is positive in CD2, CD16,CD33, CD38, and CD56.

[9] The cell line according to [1], which is negative in CD3, CD4, CD8,CD19, CD20, and CD34.

[10] The human NK cell line NK-TY2 deposited under the accession numberFERM BP-7865.

[11] An Epstein-Barr virus-infected cell of the cell line according to[1].

[12] A method for detecting an ability to control NK cell activity,which comprises the steps of:

(1) contacting a test compound with the cell line according to [1] or acell derived from the cell line; and

(2) determining an activity of said cell and comparing the activity withthat of a control.

[13] The method according to [12], wherein the cell line is the human NKcell line NK-TY2 deposited under the accession number FERM BP-7865.

[14] A method of screening for a compound having the ability to controlNK cell activity, which comprises the steps of:

(1) detecting an ability of a test compound to control NK cell activityaccording to the method of [12]; and

(2) selecting a compound that enhances or suppresses NK cell activity bycomparing the activity with that of a control.

[15] A kit for screening compounds that have an ability to control theNK cell activity, which comprises:

(a) the cell line according to [1]; and

(b) a reagent for assaying an NK cell activity.

[16] A pharmaceutical agent for controlling the NK cell activity, whichcomprises the compound selected by the screening method according to[14] as an active ingredient.

The present invention also relates to a method for controlling NK cellactivity, which comprises the step of administering the compoundselected by the screening method according to [14]. Furthermore, thepresent invention relates to a method for treating NK lymphomas, whichcomprises the step of administering the compound selected by thescreening method according to [14].

The NK cell lines of the present invention are characteristicallyEBV-negative. Such cell lines can be obtained, for example, throughlong-term passage of leukemia cells. There is no limitation on theleukemia cells so long as they are NK cells. Furthermore, the NK cellsmay be derived from any source. Preferably, cells derived from a humanlymphoma patient are used. Among others, mononuclear cells isolated fromperipheral blood of primary nasal lymphoma patients are preferredleukemia cells. A high rate of EBV infection has been reported inprimary nasal lymphomas. Thus, it is quite meaningful to clarify thecorrelation between lymphoma and EBV using an EBV-negative cell lineestablished from a primary nasal lymphoma patient. Iscove's modifiedDulbecco's medium (IMDM) culture medium (supplemented with 10% fetalbovine serum and 100 units of recombinant human IL2 (hereinafterabbreviated as “rhIL2”; Shionogi & Co. Ltd., Osaka, Japan)) or such maybe used to culture the mononuclear cells. The cells should be maintainedunder humid atmosphere with 5% CO₂ at 37° C.

In the present invention, there is no limitation on the conditions ofthe long-term passage. The presence of EBV may be confirmed, forexample, by cloning after four months, typically after six months,preferably after seven or eight months of passage. The cloning of cellscan be performed according to known techniques, such as the limitingdilution method.

Takata et al. have reported that the introduction of EBV-encoded smallRNA 1 (EBER-1) gene eliminates EBV from Burkitt's lymphoma (in theannual meeting of The Japanese Cancer Association in 1999). Theelimination of EBV from cells derived from primary nasal lymphoma may beaccomplished by the application of this method.

In general, cells, excluding T cells, having killer cell activity (NKactivity) are referred to as “NK cells”. However, some tumor cells lackthe killer cell activity (NK activity). Such cells lacking NK activitycan be confirmed as NK cells based on the presence of NK cellcharacteristic cell surface markers or cytotoxic molecules. An exemplaryexpression pattern of cell surface markers that indicates a cell to bean NK cell is shown below. These markers can be immunologically detectedby known methods using commercially available antibodies.

T cell receptor (TcR) negative CD3 negative CD56 positive NK receptorpositive

NK receptors include marker molecules such as CD94, CD158a, CD158b,CD158c, CD159, CD161, and NKG2A. When a cell is positive for at leastone of these marker molecules, it is judged to be NK receptor positive.Typically, plural markers are used as indicators.

Known cytotoxic molecules include perforin, TIA-1, Fas, Fas-L, IFN-γ,IFN-α, and Granzyme B. Likewise, a cell detected to have at least one ofthe cytotoxic molecules is judged to be cytotoxic molecule positive.Antibodies recognizing these cytotoxic molecules are commerciallyavailable. Therefore, they can be detected according to known methods,such as via the immunofluorescent antibody method.

As used herein, the term “NK cells” refers to cells either belonging togroup A or group B described below.

Group A: Group B: With killer cell activity T cell receptor (TcR)negative (NK activity) CD3 negative CD3 negative CD56 positive CD56positive T cell receptor (TcR) negative NK receptor positive TcR generearrangement negative Cytotoxic molecule positive

The presence of EBV in a cloned cell line can be confirmed, for example,by detecting the EBV genome. More specifically, for example, Southernblotting may be carried out using, as a sample, DNAs obtained bydigesting the genome of the cell line with an appropriate restrictionenzyme. Alternatively, EBV may be effectively detected by in-situhybridization using the EBV genome as the target. Furthermore, PCRmethods may be used to detect the EBV genome. The terminal repeat ofEBV, for example, may be used as a probe for Southern blotting. AnEBER-1 antisense oligonucleotide can be used as a probe in the in-situhybridization. Protocols for the Southern blotting and in-situhybridization are well known in the art.

EBV infection can be also confirmed using a technique wherein a cellsurface EBV antigen (Epstein-Barr nuclear antigen; EBNA) is detected.The detection of EBNA suggests the presence of EBV. Thus, such atechnique is an advantageous assay procedure, as a screening method toeliminate EBV-positive cells.

The NK cell lines of the present invention include the human NK cellline, NK-TY2, deposited under the accession number FERM BP-7865,internationally under the Budapest Treaty.

Depository Information:

(a) Name and Address of Depositary Institution

Name: International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology (AIST) (Previous Name: TheNational Institute of Bioscience and Human-Technology, The Agency ofIndustrial Science and Technology, The Ministry of International Tradeand Industry)Address: AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan(ZIP CODE: 305-8566)(b) Date of Deposition (Date of initial deposition): May 22, 2001(c) Accession Number: 7865 (FERM BP-7865)

NK-TY2 is an EBV-negative NK cell line established by the presentinventors through the long-term passage of mononuclear cells isolatedfrom the peripheral blood of a primary nasal angiocentric lymphoma froman EBV positive patient. The immunological phenotype of NK-TY2 is shownbelow. The NK-TY2 cell retains the phenotype of the original leukemiacell.

CD2, CD16, CD33, CD38, and CD56 positive CD3, CD4, CD8, CD19, CD20, andCD34 negative

According to analyses described herein, NK-TY2 was discovered to be acell line having the features shown below. Judging from these features,it is clear that NK-TY2 is an NK cell.

T cell receptor negative CD56 positive (TcR) TcR gene negative NKreceptor positive rearrangement (CD94 and NKG2A positive) CD3 negativeCytotoxic Positive molecule (perforin positive)

There are no limitations on the conditions for culturing the cell linesof the present invention. Thus, the cells can be cultured underarbitrary conditions which do not exterminate the cells but allow themto survive or proliferate. For example, a typical culture temperatureranges from 33° C. to 39° C., and is preferably 37° C. An IMDM mediumcontaining 3% to 10% (preferably 10%) fetal bovine serum, preferablyinactivated fetal bovine serum (i.e., fetal bovine serum heat-treated toinactivate the complements) is used as the culture medium. The cultureis maintained under an atmosphere of 5% CO₂ with 80%-120% (preferably100%) humidity.

Furthermore, there are no limitations on the conditions for storing thecell lines of the present invention. The cells can be cryopreserved, forexample, at −80° C. or in liquid nitrogen, presuspended at a celldensity of 10²-10¹⁰ cells/ml, preferably 10⁴-10⁸ cells/ml, and morepreferably 10⁶ cells/ml in a culture medium containing 10% glycerin, or10% dimethyl sulfoxide and 10% serum. Preferably, the cells arecryopreserved in liquid nitrogen, suspended at a cell density of 10⁶cells/ml in a culture medium containing 10% glycerin and 10% serum. Thecell line stored as described above can be grown again, for example, byrapidly thawing the cells in a water bath at 37° C.; adding 10 volumesof medium containing 10% serum; stirring; collecting cells bycentrifugation; and then culturing the cells in medium containing 10%serum.

The NK cell lines of the present invention are useful as a research toolto elucidate the mechanism of oncogenesis. Although the EBV genome iseliminated, the NK cell lines of the present invention remain in animmortalized state. This suggests that these cell lines have acquiredsome mutations accommodate for the function of the EBV genome. Thus, theoncogenic mechanism of lymphoma may be elucidated through analyses ofthe NK cell lines of the present invention. More specifically, cancerousmutations in lymphomas can be identified, for example, through analysesof alterations in the expression levels or mutations of knowncancer-associated genes, such as p53, p15, p16, p21, RB, bcl-2, andbcl-X, using the cells of the present invention.

For example, as revealed in the Examples, enhanced expression of c-mycand p53 was observed in NK-TY-2, an NK cell line of the presentinvention. In addition, NK-TY2 was found to be less dependent on IL2 andfetal bovine serum as compared to an EBV-positive cell line, NK-YS. Thisindicates that the cell line of the present invention, NK-TY2, may haveacquired a more malignant character, even though it is negative in EBV.Such changes at the cellular level may be important factors involved incancerous changes of lymphomas. In other words, the EBV-negative NK celllines of the present invention are useful research tools in elucidatingthe canceration mechanism of lymphomas.

As described above, EBV is suspected to play a role in lymphomas.However, particularly in Japan, while the incidence of inapparent EBVinfection is observed among many adults, only some of them developleukemia. Therefore, EBV infection alone is not responsible for thepathology of leukemia. Since the NK cell lines of the present inventionare EBV-negative, experiments can be carried out without the influenceof EBV.

Alternatively, the process of lymphoma metastasis can be studied byanalyzing endothelial damage caused by the NK cell lines of the presentinvention. More specifically, factors involved in the endothelial damagecaused by NK cells can be identified by detecting the activity ofanti-NK receptor antibodies and various adhesion molecules that inhibitendothelial damage.

In addition, NK cell lines of the present invention derived fromlymphoma patients are useful in methods for assessing or of screeningfor compounds that are effective for treating lymphoma. Cell linesestablished from patients with poor prognosis, such as primary nasallymphoma patients, are particularly useful, because they allow thescreening for therapeutic agents against diseases that are difficult totreat. Specifically, when the NK cell lines of the present inventionhave drug resistance, the drug resistance mechanism of NK lymphoma canbe elucidated using the inventive cell lines as a research tool.Chemical therapy is an important therapeutic method for the treatment oflymphomas. Thus, the elucidation of the drug resistance mechanism oflymphoma is an important area of study.

In addition, since the NK cell lines of the present invention are EBVnegative, they can be used for the isolation and expansion of EBV. Thepresence of EBV in a clinical specimen can be detected, for example, byinoculating the clinical specimen, such as blood, with the NK cell linesof the present invention and confirming the infection of EBV. EBVinfection in adults who have never been infected can cause infectiousmononucleosis. Thus, the detection of EBV in clinical specimens isuseful for diagnosing infectious mononucleosis. The infection of EBVinto cells allows their detection after the proliferation of the virus.Thus, highly sensitive measurements can be achieved by the EBV-detectingmethod using the NK cells of the present invention. Furthermore, thepresence of infectious EBV virus can be confirmed by performinginfection experiments.

Furthermore, EBV can be infected to the NK cell lines of the presentinvention. The changes in cellular character due to EBV infection can beanalyzed using an EBV-infected NK cell line. Such analysis can provideimportant information for identifying the role of EBV in NK tumor. Forexample, the involvement of EBV in the malignancy of NK tumor cells canbe studied using the cell lines of the present invention. The changes inthe character of the cell lines of the present invention may be detectedby comparing the colony formation ability in soft agar, tumor formationability in nude mice, or the sensitivity to serum concentration in theculture medium. The EBV infection to the NK cell lines of the presentinvention can be achieved by inoculating EBV with a culture of theinventive cell lines. EBV can be obtained from lysate of EBV-infectedcell lines, culture supernatant of EBV-producing cells, and such.

Furthermore, the functions of respective genes of EBV can be determinedby constructing various EBV mutants, having deletions and mutationsintroduced into the genes, and studying changes in infectivity to thecells of the present invention or characteristic changes of the cellsafter infection.

Moreover, tumor cells resistant to the NK cells can be obtained usingthe NK cell lines of the present invention. A tumor cell resistant tothe NK cells can be obtained, for example, by selecting tumor cells thatcan grow in a co-culture with the NK cells of the present invention. Themechanism of an NK cell-resistant tumor cell to evade damage by the NKcells can be examined by comparing the obtained tumor cell with an NKcell-sensitive tumor cell.

In addition, the NK cell lines of the present invention are useful as aresearch tool to discover novel physiologically active substancesproduced by the NK cells. Such physiologically active substances includeantiviral substances and antimicrobial substances. Since the NK celllines of the present invention are EBV negative, physiologically activesubstances specific to NK cells can be exclusively identified withoutcontamination of physiologically active substances whose production isinduced by EBV-derived substances.

The NK cell lines of the present invention are further useful fordetecting an ability to control NK cell activity. As used herein, thephrase “NK cell activity” refers to physiological functions of the NKcells. More specifically, “NK cell activity” comprises cell growthactivity, the activity of NK cells to produce various factors, and soon. Cytokines regulating the NK cells can be identified, for example, byexamining responses of the NK cell lines of the present invention tovarious cytokines. In addition, the cell lines of the present inventioncan be used as research tools to elucidate the signal transductionsystem mediated by the NK receptor or its functions.

For example, NK cells are known to proliferate in an IL2-dependentmanner. However, though CD25 and CD122 are predicted to be required forIL2-mediated signaling, they are not detectable in typical NK cells.Thus, the NK cell lines of the present invention can also be used toelucidate the IL2 response mechanism of NK cells because theyproliferate in an IL2-dependent manner.

The method of the present invention for detecting an ability to controlNK cell activity comprises the steps of:

(1) contacting a test compound with an NK cell line of the presentinvention or a cell derived from the NK cell line; and

(2) determining an activity of said cell and comparing the activity withthat of a control.

The above-described cell derived from the NK cell lines of the presentinvention includes mutant cell lines derived therefrom and transformantsthereof transformed with a certain gene.

According to the method for detecting an ability to control NK cellactivity of the present invention, the NK cell activity may bedetermined by measuring the activity of interest. Specifically, forexample, when the activity is one that influences the proliferationactivity of the cell, then the detection methods of the presentinvention may be performed by observing cell proliferation. Theproliferation of NK cells can be measured by monitoring [³H]-thymidineuptake.

On the other hand, various factors, such as cytokines, may be measuredto study influences on the activities of an NK cell to produce therespective factors. Alternatively, mRNAs of the respective factors maybe assayed by RT-PCR or such. Controls to be used in the presentinvention include the NK cells of the present invention that are notcontacted with the test compound, and those contacted with compoundswith known effects.

The screening of compounds that control NK cell activity can be achievedby selecting compounds for which the ability to control NK cell activitywas detected by the above-described detection method of the presentinvention. Specifically, the present invention relates to a method ofscreening for a compound that has the activity to control NK cellactivity, which comprises the steps of:

(1) contacting a test compound with an NK cell line of the presentinvention or a cell derived from the NK cell line;

(2) determining an activity of said cell, and comparing the activitywith that of a control; and

(3) selecting a compound that enhances or suppresses the NK cellactivity as compared to that of the control.

For example, compounds that suppress the proliferation or malignanttransformation of the NK cells of the present invention act not on EBVbut directly on NK cells. Conversely, when using EBV-positive cells, itis difficult to determine whether a particular compound is acting on thecells themselves or on the EBV infected to the cells. Thus, thescreening method using NK cells of the present invention is useful forselecting compounds that directly act on the NK cells.

There is no limitation on the test compounds used in the screeningmethod of the present invention. For example, cell extracts, cellculture supernatants, fermentation products of microorganisms, extractsof marine organisms, plant extracts, purified or rough-purifiedpolypeptides, non-peptidic compounds, synthetic low-molecular-weightcompounds, and natural compounds, can be screened according to thescreening method of the present invention.

NK cells play an important role in the biological defense mechanism.Thus, a compound having the activity to control NK cell activity can beused to control immunological functions. The phrase “control NK cellactivity” includes enhancement and suppression of NK cell activity. Forexample, a compound having the activity to enhance NK cell activity isexpected to potentiate immunological functions.

Conversely, a compound that suppresses an activity of the NK cell linesof the present invention is anticipated to serve as a therapeutic agentfor NK lymphoma. In particular, when a compound that suppresses theproliferation of a cell line is established from a patient with poorprognosis, such as primary nasal lymphoma patient, it may be useful as anovel therapeutic agent for lymphoma. For example, therapeutic effectsof anti-CD56 antibody, anti-CD25 antibody, or such on NK tumors can beassessed. The therapeutic effects can be evaluated using cell death asan indicator.

Compounds that can be isolated by the screening method of the presentinvention described above can be used to control NK cell activity. Suchcompounds include compounds that enhance as well as compounds thatsuppress NK cell activity. NK cells are believed to play an importantrole in the biological defense mechanism. Thus, the screening method ofthe present invention can be used to assess or isolate compounds thatcontrol the NK cell-mediated biological defense mechanism.

A kit for screening compounds that have the ability to control NK cellactivity can be constructed by combining an NK cell line required forthe screening method of the present invention and a reagent necessaryfor the detection of the NK cell activity. In the present invention, thereagent for detecting the NK cell activity can be appropriately selecteddepending on the type of activity to be used as an indicator. Forexample, when cell proliferation is to be monitored, ³H-labeledthymidine or such may be used as the reagent. Alternatively, the levelof a factor produced by the NK cell can be measured using an antibodyagainst the factor as an indicator. Furthermore, primers or probes fordetecting mRNA of the factor can be used as the reagent.

A screening kit of the present invention may further contain a culturemedium for the NK cell line of the present invention (or a cell derivedfrom the cell lines); additives, such as antibiotics and fetal bovineserum; containers for culture; and so on.

When administering an NK cell activity controlling compound obtained bythe method of the invention as a pharmaceutical for humans, othermammals, and so on, such as mice, rats, guinea-pigs, rabbits, chicken,cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees, thecompound can be directly administered or can be formulated into a dosageform using known pharmaceutical preparation methods. For example, asneeded, the compound can be taken orally, as sugar-coated tablets,capsules, elixirs, and microcapsules, or non-orally, in the form ofinjections of sterile solutions or suspensions with water or any otherpharmaceutically acceptable liquid. For example, the compound can bemixed with pharmacologically acceptable carriers or media, specifically,sterilized water, physiological saline, plant-oil, emulsifiers,suspending agents, surfactants, stabilizers, flavoring agents,excipients, vehicles, preservatives, binders, and such, in a unit doseform required for generally accepted drug implementation. The amount ofactive ingredient in the preparations makes a suitable dosage within theindicated range acquirable.

Examples of additives that can be mixed to tablets and capsules includebinders such as gelatin, corn starch, tragacanth gum, and arabic gum;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin, and alginic acid; lubricants such as magnesiumstearate; sweeteners such as sucrose, lactose, and saccharin; flavoringagents such as peppermint, Gaultheria adenothrix oil, and cherry. Whenthe unit dosage form is a capsule, a liquid carrier, such as oil, canalso be included in the above-described ingredients. Sterile compositesfor injections can be formulated according to normal drugimplementations using vehicles such as distilled water used forinjections.

Physiological saline, and isotonic liquids containing glucose and/orother adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, and non-ionic surfactants, such as Polysorbate 80™and HCO-50.

Sesame oil or soy-bean oil can be used as an oleaginous liquid and maybe used in conjunction with benzyl benzoate or benzyl alcohol assolubilizers and may be formulated with buffer, such as phosphate bufferand sodium acetate buffer; a pain-killer, such as procainehydrochloride; a stabilizer, such as benzyl alcohol and phenol; and ananti-oxidant. The prepared injection solution may be filled into asuitable ampule.

Methods well known to one skilled in the art may be used to administerthe inventive pharmaceutical compound to patients, for example, asintraarterial, intravenous, or subcutaneous injections and also asintranasal, transbronchial, intramuscular, subcutaneous, or oraladministrations. The dosage varies according to the body-weight and ageof the patient and the administration method selected; however, oneskilled in the art can readily determine an appropriate dosage. If thecompound is encodable by a DNA, the DNA can be inserted into a vectorfor gene therapy to perform the therapy. The dosage of the DNA andmethod of administration vary according to the body-weight, age, andsymptoms of the patient, though one skilled in the art can select themsuitably.

Although there are some differences according to the patient, targetorgan, symptom, and administration method, the dose of the compound isabout 0.1 mg to 100 mg per day, preferably about 1.0 mg to 50 mg perday, and more preferably about 1.0 mg to 20 mg per day, whenadministered orally to a normal adult (weight 60 kg).

When administered parenterally, in the form of an injection, to a normaladult (weight 60 kg), although there are some differences according tothe patient, target organ, symptom, and administering method, thecompound is conveniently intravenously injected at a dose of about 0.01mg to 30 mg per day, preferably about 0.1 to 20 mg per day, and morepreferably about 0.1 to 10 mg per day. Also, in the case of otheranimals, it is possible to administer an amount converted to 60 kg ofbody-weight.

All publications describing prior art cited herein are incorporated byreference herein in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts diagrams showing the IL2 dependency of NK-TY2 cells. Inthese diagrams, the added amounts of IL2 are as follows: -♦-, 1 U/ml;-▪-, 10 U/ml; and -▴-, 100 U/ml. The ordinate indicates the number ofcells (×10⁴); the abscissa indicates the days of culture (day).

FIG. 2 depicts diagrams showing the fetal bovine serum (FBS) dependencyof NK-TY2 cells. In these diagrams, the added amounts of FBS areasfollows: -♦-, 1%; -▪-, 5%; and -▴-, 10%. The ordinate indicates thenumber of cells (×10⁴); the abscissa indicates the days of culture(day).

FIG. 3 depicts a photograph of light microscopy showing NK-TY2 cellsstained by the May-Grünwald-Giemsa staining method (400-fold).

FIG. 4 depicts diagrams showing analysis results of NK-YS, NK-TY2, andTIM96 for BCL2, BCLXL, and BCL6 by flow cytometry.

FIG. 5 depicts diagrams showing analysis results of NK-YS, NK-TY2,TIM96, Raji, and K562 for p53 by flow cytometry.

FIG. 6 depicts diagrams showing analysis results of NK-YS, NK-TY2,TIM96, Raji, and Daudi for C-MYC protein by flow cytometry.

FIG. 7 depicts a photograph showing the result of chromosome analysis ofan NK-TY2 cell.

FIG. 8 depicts photographs showing the Southern blot analysis result ofthe T cell receptor gene. The upper panel shows the result obtained withthe Cβ2 probe; the lower panel shows the result obtained with the Jγprobe. In the figure, L indicates LAD cell; YS, NK-YS cell; TY2, NK-TY2cell; and J, Jurkat cell.

FIG. 9 depicts photographs showing the Southern blot analysis result ofNK-TY2 cells for the T cell receptor gene. The right panel shows theresult obtained with the Jδ1 probe; the left panel shows the resultobtained with the Jγ probe.

FIG. 10 depicts a photograph showing the Southern blot analysis resultsof EBV genome. In the figure, L indicates LAD cell; YS, NK-YS cell; TY2,NK-TY2 cell; and J, Jurkat cell.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is illustrated in detail below with reference toExamples, but is not to be construed as being limited thereto.

Example 1 Establishment of Cell Line NK-TY2

Mononuclear cells were isolated by the Ficoll-Hypaque method fromperipheral blood of a consenting patient after the crisis of leukemia.The patient was a 45 year-old female diagnosed as having primary nasalangiocentric NK lymphoma. The peripheral blood of the patient comprisedCD2+, CD3−, CD7+, and CD56+ heteromorphic lymphocytes and the patientwas EBV positive.

The isolated mononuclear cells were suspended in Iscove's modifiedDulbecco's medium (IMDM; GIBCO, Grand Island, N.Y.) supplemented with10% fetal bovine serum (FBS; Sanko Junyaku Co., Tokyo, Japan). More than90% of the cells of this cell preparation were CD56-positive. Themononuclear cells (1×10⁶ cells) were inoculated into 5 mL of IMDMsupplemented with 10% FBS containing 100 units of rhIL2 (Shionogi andCo., Osaka, Japan) in a 25 cm²-culture flask (Falcon 3013; BectonDickinson, Oxnard, Calif.). This flask was maintained under a humidifiedatmosphere with 5% CO₂ at 37° C. Half of the medium was collected twicea week, centrifuged at 1,000 rpm for 3 min in a plastic tube, and thenthe pellet was resuspended in 2.5 mL of fresh medium. The suspension wasreturned to the initial culture flask.

In the primary culture, the leukemic cells proliferated slowly duringthe first 7 months. Thereafter, the proliferation rate accelerated, andthe total number of leukemic cells reached a level of more than 1×10⁸cells at 8 month of the primary culture. At that time point, theleukemic cells showed stable proliferation. Some of these cells werecryopreserved for subsequent studies. The present inventors thenperformed cloning by the limited dilution method to establish cell lineNK-TY2. The cell doubling time of this cell line was approximately 72 hr(data not shown). The growth of NK-TY2 was IL2-dependent and wasenhanced in the presence of IL2 at a concentration of 10 U/ml or more(FIG. 1). The growth of NK-TY2 also depended on FBS and was enhanced inthe presence of 5% or more FBS (FIG. 2). NK-TY2 showed smallerdependence on IL2 and FBS as compared to NK-YS, a known NK cell line(EBV+).

Example 2 Morphological Evaluation

Cytocentrifuge smears of the peripheral blood mononuclear cells andNK-TY2 cells were stained with May-Grünwald-Giemsa for observation undera light microscope.

The result of evaluation showed that NK-YS cells had no azurophilicgranules. The cells had large nuclei with coarse chromatin andconspicuous nucleoli, and abundant basophilic cytoplasm (FIG. 3). NK-TY2cells were negative for peroxidase staining (data not shown).

Example 3 Flow Cytometric Analysis <1> (Cell Marker)

NK-TY2 cells were analyzed by single-color immunofluorescence with aflow cytometer (FACScan; Becton Dickinson and Co., Mountain View,Calif.) for the expression of surface markers. Fluoresceinisothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies usedin the experiment are shown below:

Leu5b (CD2), Leu4 (CD3), Leu3a (CD4), Leu1 (CD5), Leu9 (CD7), Leu2a(CD8), CALLA (CD10), LFA1α (CD11a), Leu15 (CD11b), LeuM5 (CD11c), LeuM7(CD13), LeuM3 (CD14), LeuM1 (CD15), Leu11 (CD16), Leu12 (CD19), Leu16(CD20), CR2 (CD21), IL2R (CD25), LeuM9 (CD33), HPCA1 (CD34), HLe1(CD45), Leu19 (CD56), Leu7 (CD57), TCR α/β, and SmIg (κ+λ) fromBecton-Dickinson;OKT6 (CD1) from Ortho Diagnostic Systems (Raritan, N.J.);B1 (CD21) from Coulter Immunology (Hialeah, Fla.);LFA3 (CD58), Fas (CD95), HP-3B1 (kp43; CD94), EB6 (CD158a), GL183(CD158b), FES172 (CD158c), Z27.3.7 (CD159), 191B8 (NKRP1A; CD161), andZ199 (NKG2A) from Immunotech (Marseilles, France); andTCR γ/δ from T Cell Sciences (Cambridge, Mass.).Throughout the flow cytometric analysis, FITC- or PE-conjugated mouseIgG was used as the negative control. Simultaneously, NK-YS, NK92, andNKL cells were analyzed in a similar manner for the expression ofsurface markers, and the results were compared.

Unconjugated antibodies against interleukin receptors were as follows:

GM-CSFR (CD116), IL4R (CD124), and IL6R (CD126) from Immunotech(Marseilles, France); and

G-CSFR (CD114), IL2Rb (CD122), IL3R (CD123), IL5R (CD125), and IL7(CD127) from PharMingen (San Diego, Calif.).

After binding these unconjugated antibodies as first antibodies to thecells, the cells were stained with PE-conjugated second antibodies andanalyzed with a flow cytometer.

The results of immunophenotyping are summarized in Table 1. The NK-TY2cells expressed CD2, CD16, CD33, CD38, and CD56 antigens but did notshow detectable levels of surface CD3, CD4, CD8, CD19, CD20, and CD34antigens. The leukemic cells from peripheral blood expressed CD2, CD7,and CD56. Thus, the phenotype of the original leukemic cells waswell-preserved in the NK-TY2 cells. The expression of CD33 seemed to beinduced during the culture process.

TABLE 1 NK-TY2 NK-YS NK92 NKL CD1 − − − − CD2 +++ +++ +++ +++ CD3 − − −− CD4 − − − − CD5 − ++ − − CD7 ++ ++ ++ ++ CD8 − − − − CD10 − − − −CD11a CD11b CD11c CD13 − − − − CD15 − − − − CD16 +++ − − ++ CD18 ++ CD19− − − − CD20 − − − − CD21 − − − − CD22 − − − − CD25 − +++ +++ +++ CD33++ − − − CD34 − − − − CD38 +++ +++ +++ +++ CD40 − − − − CD41 − − − −CD43 +++ CD44 +++ CD45 + +++ CD54 +++ CD56 +++ +++ +++ +++ CD57 − − − −CD58 +++ CD62L − − CD70 ++ CD80 ++ CD94 +++ + CD95 +++ +++ CD103 − −CD114 − CD116 − − CD117 − − CD123 − − CD124 − − CD125 − − CD126 − −CD127 − +

Example 4 Flow Cytometric Analysis <2>(Cancer- or Apoptosis-AssociatedGene

Similarly to Example 3, NK-TY2 cells were analyzed by single-colorimmunofluorescence with a flow cytometer (FACScan; Becton Dickinson andCo., Mountain View, Calif.) for the expression of oncogene oranti-oncogene products. Antibodies used are shown below:

FITC-conjugated anti-bcl2 antibody (Dakopatts);

PE-conjugated anti-p53 antibody (Pharmingen);

anti-bcl6 antibody (Dakopatts);

anti-bclXL antibody (Zymed Laboratories); and

anti-c-myc antibody (Oncogene Science)

Simultaneously, NK-YS, TIM96, Daudi, Raji, K562, and U937 cells wereanalyzed in a similar manner and the results were compared.

Each of the cell lines was stained by a standard direct or indirectconjugating method after permeabilization using Fix and Perm kits(Caltag). Specifically, cells were fixed with reagent A (fixationreagent) at room temperature (RT) and then treated with reagent B(permeabilization reagent) for 15 min. Then, cells were washed andanalyzed by a flow cytometer. According to the indirect conjugatingmethod, cells were further reacted with an FITC-conjugated secondantibody (Becton Dickinson) for 15 min, washed with PBS, and analyzedwith a flow cytometer. Throughout the flow cytometric analyses, an FITC-or PE-conjugated class matched mouse IgG, or a class matched firstantibody for indirect method was used as the negative control.

The results of the flow cytometric analyses are shown in FIG. 4 (bcl2,bclWL, and bcl6), FIG. 5 (p53), and FIG. 6 (c-myc). Similarly to theNK-YS cell line, NK-TY2 cells expressed bcl2, bclXL, and bcl6 proteins.However, the expression levels of c-myc and p53 protein were higher thanin NK-YS cells, and were comparable to Burkitt's lymphoma cell line,Daudi or Raji. These findings showed that EBV genome was easilyeliminated during culture due to the activation of the c-myc gene. Theseresults suggest that overexpression of c-myc and p53 substitute the roleof EBV genome in the NK-TY2 cell line.

Takata et al. reported the occurrence of EBV-negative clones in aculture of Akata cells derived from EBV-positive Burkitt's lymphoma.Based on the analysis of the EBV-negative clones, they revealed that EBVconfers malignant phenotypes of Burkitt's lymphoma, i.e., apoptosisresistance. The same analysis also indicated that EBV is not essentialfor in vitro growth of Burkitt's lymphoma. Indeed, EBV-negative casesaccount for 50% of Burkitt's lymphomas in Japanese patients.

In contrast, EBV is detected in all primary nasal angiocentric lymphomacases. Additionally, there is no report describing the elimination ofEBV in EBV-positive NK lymphoma cell lines during culture. Thus, EBV ispredicted to play an important role in the growth of EBV-positive NKlymphoma.

Example 5 Expression of NK Receptors

NK receptor expression in NK-TY2, NK-YS, NK92, and NKL cells wereanalyzed by indirect staining methods. NK-TY2 cells expressed CD94 andNKG2A molecules, but not CD158a, CD158b, CD158c, CD159, and CD161. Incontrast, NK-YS cells expressed low levels of CD158b, CD158c, CD94, andNKG2A (Table 2).

TABLE 2 NK-TY2 NK-YS NK92 NKL CD94 ++ + CD158a − − CD158b − +/− CD158c− + CD159 − − CD161 − − NKG2A +++ +

Example 6 Chromosomal Analysis

The cell growth of NK-TY2 cells in the logarithmic phase of cell growthwas arrested by a treatment with 0.01 mg of colcemid for 30 min beforehypotonic treatment with 0.075 M KCl. The cells were fixed with a 3:1mixture of methanol and acetic acid. The fixed cell suspension wasdropped onto a glass slide and flame-dried. The conventional G-bandingmethod was used for karyotyping. The result is shown in FIG. 7.

Seven of the 11 analyzed fresh leukemic cells in metaphase showedcomplicated karyotype of 47, X, −X, add(3) (p21), add(4) (q31), +add(6)(q13), +7, add(9) (p13), del(11) (q13q21),der(17)t(1;17)(q12;q25)ins(17;?)(q25;?), −18, +21, der(22)t(18;22)(q11;p13) (data not shown). In the analyzed cells, additionalchromosomal abnormalities such as del (X) (p22), −add(4), +add(4)(q31),−6, −add(9), +add(9)(p13), −10, add(14)(q22), −15, +18, +22,−der(22)t(18;22), and +2 mar were observed.

The G-banding analysis showed that 10 of the 10 analyzed NK-TY2 cells inmetaphase had a karyotype of 50, X, −X, +2, add(3) (p21), der(4)t(4;9)(q35;q12), del(6) (q13), +del(6), +I(7) (q10), add(9)(p22), del(11)(q13q21), der(14;16) (q10;p10), der(17)dup(17) (q21q25)t(1;17)(q12;q25), +19, +21, +mar (FIG. 7). The NK-TY2 cells preserved thecommon chromosomal abnormalities of −X, add(3)(p21), del(11) (q13q21),der(17)t(1;17) (q12;q25) observed in the original leukemic cells.

Example 7 Southern Blotting Analysis of T Cell Receptor Genes <1>

The rearrangement of the T cell receptor (hereinafter abbreviated as“TcR”) β- and γ-chain genes was evaluated according to a standardmethod. Specifically, 5 μg DNA cells was extracted from NK-TY2 accordingto a standard method, digested with EcoRI, HindIII, BamHI, or BglII,electrophoresed on a 0.6% agarose gel, and then transferred onto anitrocellulose filter. DNA extracted from human placenta was used as thenegative control. The filter was hybridized with a ³²P-labelled Cβ2 orJγ probe and washed under appropriate stringency condition, and then thebands were visualized by autoradiography. LAD, NK-YS, and Jurkat celllines were similarly analyzed, and the results were compared.

The TcR β- and γ-chain genes of the NK-TY2 cells showed a germlineconfiguration (FIG. 8). This finding agreed with the informationobtained from biopsy specimens derived from the skin lesion (data notshown). In addition, the gd_chain also showed a germline configuration.Since the sample of fresh leukemic cells was limited, the rearrangementof the TcR β- and γ-chain genes in these cells was not analyzed.

Example 8 Southern Blotting Analysis of T Cells Receptor Genes <2>

The rearrangement of TcRγ- and TcRδ-chain genes in NK-TY2 cells wasanalyzed similarly as in Example 7. Extracted DNAs were digested withEcoRI, BamHI, or KpnI. Jγ and Jδ1 were used as probes. The result isshown in FIG. 9. No rearranged band could be recognized for TcRγ andTcRδ.

Example 9 Detection of EBV Genome

DNA samples were extracted from NK-TY2 cells, EBV-transformed Blymphoblast-like cell line (LAD) as a positive control, NK-YS cells as anegative control, and Jurkat cells according to a standard method.Resulting DNAs were digested with EcoRI, PstI, BamHI, and BglIIrestriction enzymes, electrophoresed, and blotted onto nitrocellulosefilters. These filters were hybridized with a ³²P-labeled cDNA probe ofthe EBV terminal repeat, and washed under appropriate stringencycondition, and the bands were then visualized by autoradiography.

No EBV terminal repeat was detected for the NK-TY2 cells by the Southernblotting analysis (FIG. 10). In contrast, biopsy specimens werepositively stained with EBER-1 antisense oligonucleotide by the in-situhybridization method. These findings indicate that the NK-TY2 cells lostthe EBV genome during the culture process.

INDUSTRIAL APPLICABILITY

The present invention provides EBV-negative NK cell lines. The celllines of the present invention are EBV-negative, and thus provideexperimental conditions devoid of the influence of EBV. Thus, themechanism underlying the oncogenesis of NK cells due to factors otherthan EBV can be elucidated using the cell lines of the presentinvention.

A method of screening for a compound that has the ability to control NKcell activity can be established using the NK cell lines of the presentinvention. NK cells play an important role in the biological defensemechanism. Thus, a compound that controls NK cell activity can be usedto modify functions in the biological defense mechanism. Furthermore,compounds that directly act not on EBV but on NK cells can be screenedusing the EBV-negative NK cells. Moreover, novel physiologically activesubstances produced by the NK cells can be screened using the NK cellsof the present invention. In addition, the NK cell lines of the presentinvention find use in experiments for introducing foreign genes usingEBV as the vector.

1. An isolated cell of an Epstein-Barr virus (EBV)-negative naturalkiller (NK) cell line obtained by culturing EBV-positive NK lymphomacells, wherein the cell overexpresses c-myc.
 2. An isolated cell of anEBV-negative NK lymphoma cell line obtained by culturing EBV-positive NKlymphoma cells, wherein the cell overexpresses c-myc and theEBV-positive NK lymphoma cells are nasal angiocentric lymphoma cells. 3.An EBV-negative NK cell line obtained by culturing EBV-positive NKlymphoma cells, wherein the cells of the cell line overexpress c-myc. 4.A cell of the cell line designated NK-TY2 and deposited in theInternational Patent Organism Depositary of the National Institute ofAdvanced Industrial Science and Technology of Japan (AIST) under theaccession number FERM BP-7865.
 5. A cell line designated NK-TY2 anddeposited in the International Patent Organism Depositary of theNational Institute of Advanced Industrial Science and Technology ofJapan (AIST) under the accession number FERM BP-7865.
 6. A method ofmaking an EBV-negative NK cell line, the method comprising: providing apopulation of peripheral blood cells from a human subject who haslymphoma and who is EBV-positive; isolating mononuclear cells from thepopulation of peripheral blood cells, the mononuclear cells comprisingEBV-positive NK cells; maintaining the isolated mononuclear cells inculture under conditions and for a time sufficient for loss of EBV fromat least some of the NK cells to occur; selecting one or more NK cellsin which the absence of EBV is confirmed; and culturing the selected NKcell or cells, thereby providing an EBV-negative NK cell line derivedfrom an EBV-positive NK lymphoma cell, wherein the cells of the cellline overexpress c-myc.
 7. The method of claim 6, wherein the lymphomais a primary nasal lymphoma.
 8. The method of claim 6, wherein themononuclear cells are maintained in culture for at least four months. 9.The method of claim 6, wherein the cells of the EBV-negative NK cellline express CD56.
 10. An EBV-negative cell line produced by the methodof claim
 6. 11. The method of claim 6, further comprising infecting thecells of the cell line with EBV.
 12. An EBV-infected cell line producedby the method of claim
 11. 13. A method of preparing the cell of claim2, the method comprising: providing a population of peripheral bloodcells from a human subject who has nasal angiocentric lymphoma and whois EBV-positive; isolating mononuclear cells from the population ofperipheral blood cells, the mononuclear cells comprising EBV-positivenasal angiocentric NK lymphoma cells; maintaining the isolatedmononuclear cells in culture under conditions and for a time sufficientfor loss of EBV from at least some of the NK lymphoma cells to occur;and selecting an NK lymphoma cell in which the absence of EBV isconfirmed; thereby providing an isolated EBV-negative NK lymphoma cell,wherein the cell overexpresses c-myc.
 14. The method of claim 13,wherein the mononuclear cells are maintained in culture for at leastfour months.
 15. The method of claim 13, wherein the EBV-negative NKlymphoma cell expresses CD56.
 16. An isolated EBV-negative NK cellproduced by the method of claim
 13. 17. The method of claim 13, furthercomprising culturing the isolated EBV-negative NK lymphoma cell toproduce a population of EBV-negative NK lymphoma cells; infecting thepopulation of EBV-negative NK lymphoma cells with EBV to produce aplurality of EBV-infected NK lymphoma cells; and isolating anEBV-infected NK lymphoma cell from the plurality, thereby producing anisolated EBV-infected NK lymphoma cell.
 18. An isolated EBV-infectedcell produced by the method of claim
 17. 19. A method of identifying amodulator of NK cell activity, the method comprising: a. providing thecell line of claim 5; b. contacting the cell line with a test compound;and c. comparing an activity of the cell line in the presence of thetest compound to that of a control cell line in the absence of the testcompound, wherein the activity is selected from the group consisting of:i. cellular proliferation, and ii. production of a factor by the cellline; wherein a test compound that increases or decreases the activityof the cell line as compared to the control is a modulator of NK cellactivity.
 20. The method of claim 19, further comprising selecting thetest compound if it increases or decreases the activity of the cellline.
 21. A method of identifying a modulator of NK cell activity, themethod comprising: a. providing the isolated cell of claim 2; b.contacting the isolated cell with a test compound; and c. comparing anactivity of the isolated cell in the presence of the test compound tothat of a control cell in the absence of the test compound, wherein theactivity is selected from the group consisting of i. cellularproliferation, and ii. production of a factor by the isolated cell;wherein a test compound that increases or decreases the activity of theisolated cell as compared to the control is a modulator of NK cellactivity.
 22. The method of claim 21, wherein the cell line is the cellline designated NK-TY2 and deposited in the International PatentOrganism Depositary of the National Institute of Advanced IndustrialScience and Technology of Japan (AIST) under the accession number FERMBP-7865.
 23. The method of claim 21, further comprising selecting thetest compound if it increases or decreases the activity of the isolatedcell.
 24. A kit comprising the cell line of claim 3 and a reagentsuitable for assaying an activity of the cell line, wherein the activityis selected from the group consisting of (a) cellular proliferation and(b) production of a factor by the cell line.